Melanocyte-Associated T Cell Epitopes Can Function as Autoantigens for Transfer of Alopecia Areata to Human Scalp Explants on Prkdcscid Mice
2001; Elsevier BV; Volume: 117; Issue: 6 Linguagem: Inglês
10.1046/j.0022-202x.2001.01583.x
ISSN1523-1747
AutoresAmos Gilhar, Bedia Assy, Raya Shalaginov, Sima Serafimovich, Marina Landau, Richard S. Kalish,
Tópico(s)melanin and skin pigmentation
ResumoAlopecia areata is a tissue restricted autoimmune condition affecting the hair follicle, resulting in hair loss. The goal of this study was to test the hypothesis that the autoantigen of alopecia areata is melanocyte associated. Potential autoantigens were tested in the human scalp explant/Prkdscid CB-17 mouse transfer system. Scalp T cells from lesional (bald) alopecia areata scalp were cultured with antigen-presenting cells, and antigen, along with interleukin-2. The T cells were then injected into autologous lesional scalp grafts on SCID mice, and hair regrowth was measured. Hair follicle homogenate was used as an autoantigen control. T cells cultured with melanoma homogenate induced statistically significant reduction in hair growth (p <0.01 by ANOVA). HLA-A2-restricted melanocyte peptide epitopes were then tested with lesional scalp T cells from HLA-A2-positive alopecia areata patients. Melanocyte-peptide-activated T cells significantly reduced the number of hairs regrowing in two experiments with six patients (p <0.001 by ANOVA). Injected scalp grafts showed histologic and immunochemical changes of alopecia areata. The most consistent peptide autoantigens were the Gp100-derived G9-209 and G9-280 peptides, as well as MART-1 (27–35). Melanocyte peptide epitopes can function as autoantigens for alopecia areata. Multiple peptides were recognized, suggesting epitope spreading. Alopecia areata is a tissue restricted autoimmune condition affecting the hair follicle, resulting in hair loss. The goal of this study was to test the hypothesis that the autoantigen of alopecia areata is melanocyte associated. Potential autoantigens were tested in the human scalp explant/Prkdscid CB-17 mouse transfer system. Scalp T cells from lesional (bald) alopecia areata scalp were cultured with antigen-presenting cells, and antigen, along with interleukin-2. The T cells were then injected into autologous lesional scalp grafts on SCID mice, and hair regrowth was measured. Hair follicle homogenate was used as an autoantigen control. T cells cultured with melanoma homogenate induced statistically significant reduction in hair growth (p <0.01 by ANOVA). HLA-A2-restricted melanocyte peptide epitopes were then tested with lesional scalp T cells from HLA-A2-positive alopecia areata patients. Melanocyte-peptide-activated T cells significantly reduced the number of hairs regrowing in two experiments with six patients (p <0.001 by ANOVA). Injected scalp grafts showed histologic and immunochemical changes of alopecia areata. The most consistent peptide autoantigens were the Gp100-derived G9-209 and G9-280 peptides, as well as MART-1 (27–35). Melanocyte peptide epitopes can function as autoantigens for alopecia areata. Multiple peptides were recognized, suggesting epitope spreading. Alopecia areata is a tissue restricted autoimmune condition directed at the hair follicle, resulting in hair loss. Patients frequently suffer severe psychiatric consequences (Colon et al., 1991Colon E.A. Popkin M.K. Callies A.L. Dessert N.J. Hordinsky M.K. Lifetime prevalence of psychiatric disorders in patients with alopecia areata.Compr Psychiatry. 1991; 32: 245-251Abstract Full Text PDF PubMed Scopus (142) Google Scholar;Koo et al., 1994Koo J.Y. Shellow W.V. Hallman C.P. Edwards J.E. Alopecia areata and increased prevalence of psychiatric disorders.Intl J Dermatol. 1994; 33: 849-850Crossref PubMed Scopus (107) Google Scholar). This is especially true of girls and young women who become bald. The incidence of alopecia areata in the U.S.A. (Minnesota) is 20.2 per 100,000 person-years, with a lifetime risk of approximately 1.7% (Safavi et al., 1995Safavi K.H. Muller S.A. Suman V.J. Moshell A.N. Melton 3rd, L.J. Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989.Mayo Clinic Proc. 1995; 70: 628-633Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). There is no significant gender difference. The disease is often chronic with a remitting, relapsing course. Although it responds to immunosuppression, generalized immunosuppression has significant morbidity and treatment is frequently frustrating and not successful. New treatment options are essential. With evidence that alopecia areata is a T-lymphocyte-mediated autoimmune condition, it has become a model system for the study of pathogenesis and treatment of T-cell-mediated autoimmunity, and as such is a model for a host of additional T-cell-mediated autoimmune conditions. Hair loss in alopecia areata is associated with a perifollicular lymphocytic infiltrate made up primarily of CD4+ cells, with a CD8+ intrafollicular infiltrate (Todes-Taylor et al., 1984Todes-Taylor N. Turner R. Wood G.S. Stratte P.T. Morhenn V.B. T cell subpopulations in alopecia areata.J Am Acad Dermatol. 1984; 11: 216-223Abstract Full Text PDF PubMed Scopus (117) Google Scholar), along with expression of both HLA-DR and intercellular adhesion molecule 1 (ICAM-1) on the follicular epithelium (Messenger and Bleehen, 1985Messenger A.G. Bleehen S.S. Expression of HLA-DR by anagen hair follicles in alopecia areata.J Invest Dermatol. 1985; 85: 569-572Crossref PubMed Scopus (90) Google Scholar;Khoury et al., 1988Khoury E.L. Price V.H. Greenspan J.S. HLA-DR expression by hair follicle keratinocytes in alopecia areata: evidence that it is secondary to the lymphoid infiltration.J Invest Dermatol. 1988; 90: 193-200Abstract Full Text PDF PubMed Google Scholar). It is presumed that the follicular expression of DR and ICAM-1 is induced by interferon-γ (INF-γ). Lesional scalp from alopecia areata patients grafted onto nude mice regrows hair coincident with a loss of infiltrating lymphocytes from the graft (Gilhar and Krueger, 1987Gilhar A. Krueger G.G. Hair growth in scalp grafts from patients with alopecia areata and alopecia universalis grafted onto nude mice.Arch Dermatol. 1987; 123: 44-50Crossref PubMed Scopus (68) Google Scholar). Alopecia areata responds to immunosuppressive doses of systemic steroids. The condition also has an association with additional autoimmune processes such as autoimmune thyroiditis (Milgraum et al., 1987Milgraum S.S. Mitchell A.J. Bacon G.E. Rasmussen J.E. Alopecia areata, endocrine function, and autoantibodies in patients 16 years of age or younger.J Am Acad Dermatol. 1987; 17: 57-61Abstract Full Text PDF PubMed Scopus (68) Google Scholar) and vitiligo (Shellow et al., 1992Shellow W.V. Edwards J.E. Koo J.Y. Profile of alopecia areata: a questionnaire analysis of patient and family.Intl J Dermatol. 1992; 31: 186-189Crossref PubMed Scopus (130) Google Scholar). Circulating autoantibodies to follicular structures are present in alopecia areata, but they are also reported in normal controls (Tobin et al., 1997Tobin D.J. Hann S.K. Song M.S. Bystryn J.C. Hair follicle structures targeted by antibodies in patients with alopecia areata.Arch Dermatol. 1997; 133: 57-61Crossref PubMed Google Scholar), and there is no consistent pattern of reactivity of these antibodies. Furthermore, it is not possible to transfer alopecia areata by injection of IgG into human skin explants on nude mice (Gilhar et al., 1992Gilhar A. Pillar T. Assy B. David M. Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice.Br J Dermatol. 1992; 126: 166-171Crossref PubMed Scopus (51) Google Scholar). Severe combined immunodeficiency (SCID) mice do not reject human lymphocytes or skin. SCID mice grafted with human skin and injected with human lymphocytes can be used to study the immunopathogenesis of human skin conditions, and have been used to demonstrate the role of lymphocytes in induction (Boehncke et al., 1996Boehncke W.H. Dressel D. Zollner T.M. Kaufmann R. Pulling the trigger on psoriasis.Nature. 1996; 379: 777Crossref PubMed Scopus (107) Google Scholar;Wrone-Smith and Nickoloff, 1996Wrone-Smith T. Nickoloff B.J. Dermal injection of immunocytes induces psoriasis.J Clin Invest. 1996; 98: 1878-1887Crossref PubMed Scopus (381) Google Scholar) and maintenance (Gilhar et al., 1997Gilhar A. David M. Ullman Y. Berkutski T. Kalish R.S. T-Lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.J Invest Dermatol. 1997; 109: 283-288Crossref PubMed Scopus (114) Google Scholar) of psoriasis lesions. We have since extended this model to the use of human scalp explants on SCID mice to study alopecia areata. We have shown that it is possible to transfer alopecia areata to human scalp explants on SCID mice by the injection of scalp-infiltrating T lymphocytes (Gilhar et al., 1998Gilhar A. Ullmann Y. Berkutzki T. Assy B. Kalish R.S. Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocyte injections.J Clin Invest. 1998; 101: 62-67Crossref PubMed Scopus (224) Google Scholar). It was necessary to first activate the T lymphocytes in vitro by culture with hair follicle homogenate in the presence of antigen-presenting cells (APC). Culture with nonfollicular scalp homogenate did not induce hair loss (Gilhar et al., 1999Gilhar A. Shalaginov R. Assy B. Serafimovich S. Kalish R.S. Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice.J Invest Dermatol Symp Proc. 1999; 4: 207-210Abstract Full Text PDF PubMed Scopus (70) Google Scholar). Thus, alopecia areata is mediated by T lymphocytes that recognize a hair follicle antigen. We have since found that optimal transfer of hair loss requires both CD4+ and CD8+ T cells (Gilhar et al.,A. Gilhar, M. Landau, B. Assy, R. Shalaginov, S. Serafimovich, R.S. Kalish: Alopecia areata is mediated by cooperation between CD4+ and CD8+ T-lymphocytes: transfer to human scalp explants on Prkdcscid mice, Arch Dermatol in pressGoogle Scholar). Depletion of either CD8+ T cells (McElwee et al., 1996McElwee K.J. Spiers E.M. Oliver R.F. In vivo depletion of CD8+ T-cells restores hair growth in the DEBR model for alopecia areata.Br J Dermatol. 1996; 135: 211-217Crossref PubMed Scopus (92) Google Scholar) or CD4+ T cells (McElwee et al., 1999McElwee K.J. Spiers E.M. Oliver R.F. Partial restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4+ T cells.Br J Dermatol. 1999; 140: 432-437Crossref PubMed Scopus (71) Google Scholar) can reverse alopecia areata in the Dundee experimental bald rat, supporting a synergy or cooperation between CD8+ and CD4+ T cells. Inflammatory T cells of alopecia areata are cytotoxic and possess both the Fas/Fas ligand and granzyme B cytotoxic mechanisms (Bodemer et al., 2000Bodemer C. Peuchmaur M. Fraitaig S. Chatenoud L. Brousse N. de Prost Y. Role of cytotoxic T cells in chronic alopecia areata.J Invest Dermatol. 2000; 114: 112-116Crossref PubMed Scopus (106) Google Scholar). It is proposed that the CD4+ T cells provide help for the effector function of the CD8+ T cells. It is hypothesized that the autoantigen of alopecia areata is melanocyte derived (Paus et al., 1994Paus R. Slominski A. Czarnetzki B.M. Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb?.Yale J Biol Med. 1994; 66: 541-554Google Scholar). The initial basis for this is the frequent clinical observation that with disease activity pigmented hairs are lost preferentially to nonpigmented (e.g., white) hairs. Furthermore, with regrowth, there is a tendency for the initial regrowing hairs to be white. Melanocytes are a significant component of the hair bulb, which is the site of immunologic attack. Furthermore, there is an association of alopecia areata with vitiligo (Shong and Kim, 1991Shong Y.K. Kim J.A. Vitiligo in autoimmune thyroid disease.Thyroidology. 1991; 3: 89-91PubMed Google Scholar;Shellow et al., 1992Shellow W.V. Edwards J.E. Koo J.Y. Profile of alopecia areata: a questionnaire analysis of patient and family.Intl J Dermatol. 1992; 31: 186-189Crossref PubMed Scopus (130) Google Scholar), and hair bulb melanocytes in alopecia areata show both histologic and ultrastructural abnormalities (Tobin et al., 1990Tobin D.J. Fenton D.A. Kendall M.D. Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata.J Invest Dermatol. 1990; 94: 803-807Abstract Full Text PDF PubMed Google Scholar). It is possible to induce lymphocyte-mediated alopecia in mice immunized against melanocyte-associated (e.g., melanoma) antigens (Becker et al., 1996Becker J.C. Varki N. Brocker E.B. Reisfeld R.A. Lymphocyte-mediated alopecia in C57Bl/6 mice following successful immunotherapy for melanoma.J Invest Dermatol. 1996; 107: 627-632Crossref PubMed Scopus (33) Google Scholar). There is a large literature on melanocyte-associated T cell epitopes generated by the search for melanoma-associated antigens (Bakker et al., 1995Bakker A.B. Schreurs M.W. Tafazzul G. de Boer A.J. Kawakami Y. Adema G.J. Figdor C.G. Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.Intl J Cancer. 1995; 62: 97-102Crossref PubMed Scopus (114) Google Scholar;Rivoltini et al., 1995Rivoltini L. Kawakami Y. Sakaguchi K. et al.Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.J Immunol. 1995; 154: 2257-2265PubMed Google Scholar;Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar;Salgaller et al., 1996Salgaller M.L. Marincola F.M. Cormier J.N. Rosenberg S.A. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.Cancer Res. 1996; 56: 4749-4757PubMed Google Scholar;Fleischhauer et al., 1997Fleischhauer K. Tanzarella S. Russo V. Sensi M.L. van der Bruggen P. Bordignon C. Traversari C. Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones.J Immunol. 1997; 159: 2513-2521PubMed Google Scholar;Salazar-Onfray et al., 1997Salazar-Onfray F. Nakazawa T. Chhajlani V. et al.Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.Cancer Res. 1997; 57: 4348-4355PubMed Google Scholar;Bettinotti et al., 1998Bettinotti M.P. Kim C.J. Lee K.H. et al.Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy.J Immunol. 1998; 161: 877-889PubMed Google Scholar;Kittlesen et al., 1998Kittlesen D.J. Thompson L.W. Gulden P.H. et al.Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development.J Immunol. 1998; 160: 2099-2106PubMed Google Scholar). Most interest has focusedonpeptidespresentedby HLA-A2, which is present in approximately half the population 1Antigen frequencies from Histocompatibility Testing 1980. Los Angeles: UCLA Tissue Typing Laboratory, 1980.1Antigen frequencies from Histocompatibility Testing 1980. Los Angeles: UCLA Tissue Typing Laboratory, 1980.. HLA-A2 may also be associated with alopecia areata (Hordinsky et al., 1984Hordinsky M.K. Hallgren H. Nelson D. Filipovich A.H. Familial alopecia areata. HLA antigens and autoantibody formation in an American family.Arch Dermatol. 1984; 120: 464-468Crossref PubMed Scopus (43) Google Scholar). Many of the antigenic peptides are derived from melanosome-associated proteins such as gp100, MART-1/Melan-A, melanocortin 1 receptor (MC1R), or tyrosinase, and are present in both normal melanocytes and melanomas. MAGE antigens tend to be preferentially expressed on melanoma cells, and are less likely to function as autoantigens for alopecia areata. The goal of this study was to test the hypothesis that melanocyte-associated antigens can function as autoantigens to induce hair loss in alopecia areata. For this purpose, HLA-A2-positive patients with alopecia areata were selected for studies of HLA-A2-restricted melanocyte peptide epitopes. Scalp T cells were cultured with autologous APC and either hair follicle homogenate (positive control) or melanocyte T cell epitopes. The cells were then transferred to autologous scalp explants on SCID mice, inducing hair loss. Ten patients with either alopecia totalis or severe alopecia areata were studied. Specimens from four patients were used for experiments with melanoma homogenate, and specimens from six patients were used for experiments with melanocyte-associated peptides. HLA-A2-positive alopecia areata patients were selected for the peptide component of this study. The four donors used for the melanoma homogenate experiment were not HLA typed. Severe alopecia areata was defined as large areas of alopecia with small residual areas of hair. These patients would be categorized as S4 (76%-99% hair loss) by the alopecia areata investigational assessment guidelines (Olsen et al., 1999Olsen E. Hordinsky M. McDonald-Hull S. Price V. Roberts J. Shapiro J. Stenn K. Alopecia areata investigational assessment guidelines.J Am Acad Dermatol. 1999; 40: 242-246Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Duration, age, sex, and clinical characteristics of all donors are listed in Table I. There were no other preselection criteria, and data on these 10 patients were previously unpublished. Informed consent was obtained after the nature and possible consequences of the studies were explained. Protocols were approved by the Institutional Review Board. None of the patients had any therapy in the 60 d before obtaining the scalp biopsy specimens. HLA-A2 expression was determined by microlymphocytotoxicity assay (Immunology Allergy and AIDS Institute, Tissue Typing Laboratory, B. Rappaport Faculty of Medicine, Technion and Rambam Medical Center, Haifa).Table IClinical summary of alopecia areata patientsPatientAgeSexAlopecia subtypeDurationA31FSevereaSevere alopecia areata is defined as large areas of alopecia with small residual areas of hair, or nearly complete alopecia totalis (76%-99% hair loss). Donors A-D were studied in the melanoma homogenate experiment. Donors 1–6 are HLA-A2 positive and were studied in the melanomo peptide experiments.8 yB32MSevere4 yC26FSevere10 yD26FSevere10 y123MSevere7 y239FSevere2 y336MSevere2 y432MTotalis6 y522FSevere10 y631MTotalis10 ya Severe alopecia areata is defined as large areas of alopecia with small residual areas of hair, or nearly complete alopecia totalis (76%-99% hair loss). Donors A-D were studied in the melanoma homogenate experiment. Donors 1–6 are HLA-A2 positive and were studied in the melanomo peptide experiments. Open table in a new tab Punch biopsies (2 mm) from involved scalp were obtained from each patient. Four biopsies from each patient were used for the isolation of cutaneous T cells. Two biopsies were snap-frozen in liquid nitrogen for immunoperoxidase staining. One frozen biopsy was sectioned vertically, and the other was sectioned horizontally. An additional biopsy from each patient was submitted for routine histologic examination with hematoxylin and eosin and horizontally sectioned in its entirety. Punch biopsies (2 mm) were grafted to SCID mice in sets of three biopsies per mouse, one mouse per group per subject. CB-17 Prkdcscid (SCID) mice (Charles River, U.K.), 2–3 mo of age, were used in this study. The mice were raised in the pathogen-free animal facility of the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology. Animal care and research protocols were in accordance with institutional guidelines. Graft transplantation to SCID mice was performed as previously described (Gilhar and Krueger, 1987Gilhar A. Krueger G.G. Hair growth in scalp grafts from patients with alopecia areata and alopecia universalis grafted onto nude mice.Arch Dermatol. 1987; 123: 44-50Crossref PubMed Scopus (68) Google Scholar;Gilhar et al., 1988Gilhar A. Pillar T. Etzioni A. The effect of topical cyclosporin on the immediate shedding of human scalp hair grafted onto nude mice.Br J Dermatol. 1988; 119: 767-770Crossref PubMed Scopus (23) Google Scholar,Gilhar et al., 1998Gilhar A. Ullmann Y. Berkutzki T. Assy B. Kalish R.S. Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocyte injections.J Clin Invest. 1998; 101: 62-67Crossref PubMed Scopus (224) Google Scholar). Each 2 mm graft was inserted through an incision in the skin into the subcutaneous tissue over the lateral thoracic cage of each mouse, and covered with a standard Band Aid dressing. The dressing was removed on day 7. Four punch biopsies from each patient were used for T cell isolation. Tissue-infiltrating lymphocytes were isolated from scalp punch biopsies with the use of collagenase (Sigma, St Louis, MO), as described previously (Gilhar et al., 1998Gilhar A. Ullmann Y. Berkutzki T. Assy B. Kalish R.S. Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocyte injections.J Clin Invest. 1998; 101: 62-67Crossref PubMed Scopus (224) Google Scholar). PBMC were isolated from heparinized whole blood by centrifugation over Hypaque 1077 (Amersham Pharmacia Biotech, Uppsala, Sweden). Anagen hair follicles were isolated under a stereodissecting microscope from normal scalp biopsies obtained from healthy subjects who underwent hair transplantation procedures. The follicles were homogenized and processed as previously described (Gilhar et al., 1998Gilhar A. Ullmann Y. Berkutzki T. Assy B. Kalish R.S. Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocyte injections.J Clin Invest. 1998; 101: 62-67Crossref PubMed Scopus (224) Google Scholar). Peptides were selected based on identification in the literature as HLA-A2-restricted melanocyte (e.g., melanoma) associated T cell epitopes. The following peptides were commercially synthesized (Chiron Technologies, Raleigh, NC) with free amino and carboxylic acid termini: Gp100/G9-154, KTWGQYWQV (Bakker et al., 1995Bakker A.B. Schreurs M.W. Tafazzul G. de Boer A.J. Kawakami Y. Adema G.J. Figdor C.G. Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.Intl J Cancer. 1995; 62: 97-102Crossref PubMed Scopus (114) Google Scholar;Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar;Salgaller et al., 1996Salgaller M.L. Marincola F.M. Cormier J.N. Rosenberg S.A. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.Cancer Res. 1996; 56: 4749-4757PubMed Google Scholar); Gp100/G9-209, ITDQVPFSV (Bakker et al., 1995Bakker A.B. Schreurs M.W. Tafazzul G. de Boer A.J. Kawakami Y. Adema G.J. Figdor C.G. Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.Intl J Cancer. 1995; 62: 97-102Crossref PubMed Scopus (114) Google Scholar;Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar;Salgaller et al., 1996Salgaller M.L. Marincola F.M. Cormier J.N. Rosenberg S.A. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.Cancer Res. 1996; 56: 4749-4757PubMed Google Scholar); Gp100/G9-280, YLEPGPVTA (Bakker et al., 1995Bakker A.B. Schreurs M.W. Tafazzul G. de Boer A.J. Kawakami Y. Adema G.J. Figdor C.G. Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.Intl J Cancer. 1995; 62: 97-102Crossref PubMed Scopus (114) Google Scholar;Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar;Salgaller et al., 1996Salgaller M.L. Marincola F.M. Cormier J.N. Rosenberg S.A. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.Cancer Res. 1996; 56: 4749-4757PubMed Google Scholar); MC1R 291, AIIDPLIYA (Salazar-Onfray et al., 1997Salazar-Onfray F. Nakazawa T. Chhajlani V. et al.Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.Cancer Res. 1997; 57: 4348-4355PubMed Google Scholar); MC1R 244, TILLGIFFL (Salazar-Onfray et al., 1997Salazar-Onfray F. Nakazawa T. Chhajlani V. et al.Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.Cancer Res. 1997; 57: 4348-4355PubMed Google Scholar); MC1R 283, FLALIICNA (Salazar-Onfray et al., 1997Salazar-Onfray F. Nakazawa T. Chhajlani V. et al.Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.Cancer Res. 1997; 57: 4348-4355PubMed Google Scholar); MART-1 (27–35), AAGIGILTV (Bettinotti et al., 1998Bettinotti M.P. Kim C.J. Lee K.H. et al.Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy.J Immunol. 1998; 161: 877-889PubMed Google Scholar;Rivoltini et al., 1995Rivoltini L. Kawakami Y. Sakaguchi K. et al.Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.J Immunol. 1995; 154: 2257-2265PubMed Google Scholar); and tyrosinase, AFLPWHRFL (Kang et al., 1995Kang X. Kawakami Y. el-Gamil M. et al.Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes.J Immunol. 1995; 155: 1343-1348PubMed Google Scholar). Peptides were negative for endotoxin by the Limulus Amebocyte Lysate method (Bactochem, Nes Ziona, Israel). MeWo cells were obtained from Dr. Z. Smetana (Central Virology Laboratory, Chaim Sheba Medical Center, Tel Hashomer, Israel) (Sauvaigo et al., 1986Sauvaigo S. Fretts R.E. Riopelle R.J. Lagarde A.E. Autonomous proliferation of MeWo human melanoma cell lines in serum-free medium: secretion of growth-stimulating activities.Int J Cancer. 1986; 37: 123-132Crossref PubMed Scopus (24) Google Scholar). They were propagated in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 1% sodium pyruvate, plus penicillin/streptomycin. Lymphocytes were plated at 1 × 105 cells per well in RPMI-FCS, along with irradiated (5000 R) PBMC (1 × 105 cells per well) in 24 plate wells (Greiner, Lake Mary, FL), as feeders. After 3 d of culture, recombinant interleukin-2 (rIL-2, 10 U per ml; Genzyme Diagnostics, San Carlos, CA) was added. Every 7 d the T cells were re-stimulated with feeders, for a total of 21 d culture. As indicated for experimental groups, lymphocytes were stimulated with either follicular homogenate (10 µg per ml) or peptide (10 µg per ml), along with the feeder cells at each stimulation. Phenotype of the cultured T cell lines was determined by immunocytochemistry on cytospin preparations, using procedures we have previously published (Gilhar et al., 1997Gilhar A. David M. Ullman Y. Berkutski T. Kalish R.S. T-Lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.J Invest Dermatol. 1997; 109: 283-288Crossref PubMed Scopus (114) Google Scholar). This approach was preferred over cytofluorograph analysis because of the small numbers of cells available. The following monoclonal antibodies were used: anti-CD3 FITC (clone SK 7, Becton Dickinson), anti-CD4 (IgG2a, S3.5), anti-CD8 (IgG2a, 3B5) (Caltag, Burlingame, CA), anti-HLA-DR (IgG2a, DK22, Dako, Glostrup, Denmark). Subtype matched mouse IgG-FITC (Becton Dickinson) was used as a negative control. The SCID mice bearing grafts of lesional scalp were divided into groups as indicated for each experiment. Between the twenty-third and thirtieth days, the grafts were injected intradermally (0.1 ml) with autologous lymphocytes as noted for each experiment. On day 90 biopsies were obtained for immunohistochemical and histologic analysis. Both the percentage of grafts with hair and the number of hairs per graft were recorded. Staining was performed as reported previously (Gilhar et al., 1998Gilhar A. Ullmann Y. Berkutzki T. Assy B. Kalish R.S. Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocyte injections.J Clin Invest. 1998; 101: 62-67Crossref PubMed Scopus (224) Google Scholar). Monoclonal antibo
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